One of the most fundamental processes in cell biology is the division of a mother cell into two daughter cells, resulting in the faithful partitioning of the cytoplasm and genetic material into two equal parts. While this process of cytokinesis clearly involves the contractile proteins actin and myosin, which accumulate in the furrow as a contractile ring, virtually nothing is understood at the molecular level about any of the key issues surrounding this process. How much of the actin and myosin concentrates in the furrow region of the dividing cell? How do these proteins know to concentrate there at exactly the right place and at the right time in the cell cycle? What is the detailed molecular architecture of the contractile ring? What turns on the contractile event and what terminates it? What limits the velocity of the furrowing process? How does the contractile ring get out of the way in the final stages of cytokinesis so as not to inhibit the necessary membrane fusion event to yield two independent daughter cells? What role, if any, does traction on a surface play in the cell division process? These are just some of the many questions that need to be answered in molecular terms. Key to a long term undertaking to solve this problem is the judicious choice of a model system. Dictyostelium discoideum is a model of choice because it's behavioral characteristics during mitosis and cytokinesis are extremely similar to mammalian cells. It is, however, a much simpler eukaryotic cell with only 1 percent of the genome size of a mammalian cell. Furthermore, it has single copy genes, it is haploid, molecular genetic approaches have been well worked out, and homologous recombination is very efficient. In addition, a wealth of information about a key player in cytokinesis, the myosin molecule, is already available. This proposal represents a long-term commitment to study cytokinesis in molecular detail. To lay crucial groundwork towards this long-term goal, detailed characterization of the dynamic relocalization of myosin as cells enter mitosis and proceed through cytokinesis will be accomplished. The structure/function relationships of myosin in regard to its in vivo role in cytokinesis will be examined. The goal of identifying other key genes that are involved and isolating all of the essential proteins will be pursued. Finally, synchronization of cytokinesis in populations of Dictyostelium cells for biochemical studies will be attempted.